PROJECT SUMMARY: Reducing incidence of HIV-1 by developing a vaccine is a high priority NIH research goal, and strategies to enhance high affinity, protective antibody responses are critically needed to achieve this goal. The microbiome is known to affect overall immunity, and therefore, strategies to harness the microbiome to increase effective antibody responses to HIV-1 vaccination should be explored. The overall objective for this application is to analyze genetically and functionally how adjuvanting a vaccine by microbiome manipulation affects the vaccine-specific B cell responses as well as homeostatic commensal responses. We will exploit previously collected blood, bone marrow and mucosal samples from an HIV-1 vaccination trial in rhesus macaques consisting of SIV (gag p55) and HIV (gp140) DNA + HIV gp140 trimer protein in the presence or absence of probiotic therapy, which was used as an adjuvant to enhance immunogenicity through microbiome alteration. In this highly novel, exploratory R21, we hypothesize that enhancing the microbiome by using probiotics as an adjuvant will boost affinity maturation of the vaccine- specific B cells as well as alter anti-commensal and commensal/HIV-1 cross-reactive responses. We will determine the effects on B cell affinity maturation by sorting gp140-specific B cells and nex-gen sequencing (NGS) B cell receptors (BCRs) to analyze lineages over time. We will determine the effects on anti-commensal antibody responses in mucosa and circulating blood using whole bacteria ELISA. We will determine the effects of microbiome alteration on pre-existing HIV-1 cross-reactive B cells by separately sorting these gp140-specific B cells at pre-vaccination time points and tracing their lineage development in our NGS BCR libraries. After conclusion of this project, we will know the extent to which microbiome manipulation can adjuvant vaccine responses and affect anti-commensal homeostatic immunity in the context of vaccine efficacy. These data will provide critical information on the role of the microbiome in adjuvant design and vaccine responses that may be harnessed for future studies to improve vaccine trials. Furthermore, these data will provide the rationale for assessment of ongoing clinical trials involving microbiome alteration.